Involvement of MST1/mTORC1/STAT1 activity in the regulation of B‐cell receptor signalling by chemokine receptor 2

Abstract Background CCR2 is involved in maintaining immune homeostasis and regulating immune function. This study aims to elucidate the mechanism by which CCR2 regulates B‐cell signalling. Methods In Ccr2‐knockout mice, the development and differentiation of B cells, BCR proximal signals, actin movement and B‐cell immune response were determined. Besides, the level of CCR2 in PBMC of SLE patients was analysed by bioinformatics. Results CCR2 deficiency reduces the proportion and number of follicular B cells, upregulates BCR proximal signalling and enhances the oxidative phosphorylation of B cells. Meanwhile, increased actin filaments aggregation and its associated early‐activation events of B cells are also induced by CCR2 deficiency. The MST1/mTORC1/STAT1 axis in B cells is responsible for the regulation of actin remodelling, metabolic activities and transcriptional signalling, specific MST1, mTORC1 or STAT1 inhibitor can rescue the upregulated BCR signalling. Glomerular IgG deposition is obvious in CCR2‐deficient mice, accompanied by increased anti‐dsDNA IgG level. Additionally, the CCR2 expression in peripheral B cells of SLE patients is decreased than that of healthy controls. Conclusions CCR2 can utilise MST1/mTORC1/STAT1 axis to regulate BCR signalling. The interaction between CCR2 and BCR may contribute to exploring the mechanism of autoimmune diseases.

2020M682419; HUST Academic Frontier Youth Team, Grant/Award Number: 2018QYTD10 MST1, mTORC1 or STAT1 inhibitor can rescue the upregulated BCR signalling. Glomerular IgG deposition is obvious in CCR2-deficient mice, accompanied by increased anti-dsDNA IgG level. Additionally, the CCR2 expression in peripheral B cells of SLE patients is decreased than that of healthy controls. Conclusions: CCR2 can utilise MST1/mTORC1/STAT1 axis to regulate BCR signalling. The interaction between CCR2 and BCR may contribute to exploring the mechanism of autoimmune diseases.

K E Y W O R D S
autoimmune diseases, B-cell receptor, CCR2, MST1, mTORC1

BACKGROUND
Exogenous antigens can induce B-cell receptor (BCR) activation and signal cascade. 1 BCR is coupled to disulphide heterodimers CD79a and CD79b non-covalently, and then the proximal signalling molecules are activated. 2 Syk can be recruited to phosphorylated immunoreceptor tyrosine-based activation motifs, then activate BTK and PLCγ2. 3 CD19 recruits BTK by activating PI3K, which is critical for amplifying downstream signalling. In different stages of BCR clustering, cytoskeleton actin can be targeted by B-cell signalling to undergo characteristic remodelling, F-actin accumulates near BCR microclusters in a polarised manner. [4][5][6] In addition to antibody secretion, B cells perform multiple functions in the immune system, including antigen presentation and cytokine secretion. 7 Mammalian target of rapamycin (mTOR) can facilitate B-cell activation and autoantibody production, 8 and the clinical efficacy of rapamycin in the treatment of rheumatism has been proved. 10,9 During B-cell activation, MST1 functions as a molecular brake to balance immune tolerance, its depletion leads to hypergammaglobulinemia in mice. 11 MST1-deficient mice have decreased marginal zone (MZ) B cells and downregulated BCR clustering. 12 Besides, clinical efficacy of JAK inhibitor supported the involvement of JAK/STAT pathway in the evolution of systemic lupus erythematosus (SLE). 13 Chemokine (C-C motif) receptor 2 (CCR2) interacts with its ligand CCL2 at different expression levels to induce the recruitment of chemotactic cells to tissues, maintain immune homeostasis and regulate autoimmunity. [14][15][16] The decreased CCR2 expression in T cells is correlated to diseased activity of SLE, 17 and CCR2 is involved in recruiting basophil to induce skin lesions in SLE patients. 18 Functional and constitutive CCR2 can be expressed in B cells, 19 and in the case of parasitic infection, B-cell growth was enhanced in CCR2 -/mice. 20 CCR2 and CCL2 have been reported as negative homing regulators. 21 However, the mechanism by which CCR2 regulates B-cell signalling in immune disorders remains unclear.
Therefore, we used CCR2-deficient mice to explore whether B-cell signalling or related biologic activity will be affected by the absence of CCR2, and attempted to elucidate the molecular mechanisms involved.

Animals
Ccr2 knockout (KO) mice, wild type (WT) and μMT mice on C57BL/6J background were purchased from Jackson laboratory and bred in a SPF animal department. Ccr2-KO mice were hybridised with WT mice to obtain same background heterozygous Ccr2 littermates to be used as controls. Mice between 6-and 8-week-old were used. Animal experimentation was reviewed and approved by the Institutional Animal Care and Ethics Committee of Animal Experimentation of Tongji Medical College.

Cell isolation and purification
The spleen of mouse was grinded and gradient centrifuged by Ficoll-Hypaque solution for 20 min (room temperature, brake = 5) to obtain mononuclear cells. B cells were then purified from splenic mononuclear cells by incubation of anti-Thy-1 (105310, BioLegend) and guinea pig complement (C300-0500, Rockland Immunochemicals) for 30 min (37 • C), followed by incubation for 1 h to remove adherent cells, as described previously. 22 Unilateral femoral and tibial bone marrow (BM) cavities were rinsed twice with 10 ml HBSS medium containing foetal bovine serum, after centrifugation, cells were lysed with Red Cell Lysis Buffer (RT122-02, Tiagen) for 1-2 min, then washed and filtered. Ice-cold PBS (5 ml) was injected into the mice peritoneal cavity and gently massaged for 1-2 min, fluid was collected and centrifuged to get cells.
For human blood sample, serum was removed after centrifugation at 3000 rpm for 10 min, the cell precipitate was suspended with PBS, and then slowly added into the tube of Ficoll-Hypaque solution (ratio of 1:1). After centrifugation at 2000 rpm for 20 min (brake = 5), peripheral blood mononuclear cells (PBMC), granulocytes (PG), and white blood cells (PW) were obtained according to different densities. For PBMC of SLE patients, after incubation with anti-B220, cells were fixed and permeabilised, and then incubated with anti-CCR2 (ab203128, Abcam).
Samples were analysed by Attune™ NxT sonic focused flow cytometer (Thermo Fisher) and FlowJo software (BD Biosciences).
Representative images were taken and analysed for mean fluorescence intensity (MFI), B-cell contact area (under interference reflection microscopy, IRM), and colocalisation using NIS elements AR 5.01 software (Nikon). For CFm and TIRFm assay, 60× or 100× oil lens of microscopy were used respectively. Image resolution is 2048 × 2048. Data from three independent experiments using more than 30-50 individual cells were included for each parameter.

In vitro specific inhibition
Purified

Seahorse analysis
Cell metabolism were detected by using Seahorse XFe24 cell metabolism analyser (XFe24, Agilent Technologies).

Calcium flux assay
Purified splenic B cells (5 × 10 6 ) were labelled with 0.5 μM calcium-sensitive dye Fluo-4 AM (S1060, Beyotime) in Ca 2+ free balanced salt solution for 25 min (37 • C), and then stained with anti-B220 Ab for 30 min. Using a LSRII flow cytometer (BD Biosciences), a baseline fluorescence intensity was recorded for the first 30 s, then cells were immediately stimulated with 10 μg/ml pre-warmed F(ab') 2 anti-mouse Ig (M + G) for the next 270 s to analyse Ca 2+ flux kinetics.

Scanning electron microscopy (SEM)
Incubate poly-D-lysine coated sterile coverslips with 10 μg/ml F(ab') 2 -Ig (M + G) at 37 • C for 3 h. Purified B cells (3 × 10 6 /ml) were added gently onto the coverslips for 10 min, then 400 μl 2.5% glutaraldehyde were added to terminate the stimulation and fix the cells for 20 min (on ice). Micrographs were captured using an Ultra-high Resolution SEM (SU8010, HITACHI). Cellular filopodia expansion was imaged and the number and length of filopodia were quantified using ImageJ software (Bethesda).

Immunisation and enzyme-linked immunosorbent assay (ELISA)
Mice were injected intraperitoneally (i.p.) with 40 μg 4-hydroxy-3-nitrophenylacetyl conjugated keyhole limpet haemo-cyanin (NP-KLH) (N-5060-25, Biosearch Technologies), supplemented with Freund's incomplete adjuvant (S6322-1VL, Sigma Aldrich). Fourteen days post-prime, mice were boosted with the same reagents. Five days later, mice were euthanised and spleens were harvested. FCM was performed to test the proportion and cell number of B-cell subsets. ELISA was performed using NP30-bovine serum albumin (BSA)-coated plates, according to the manufacturer's information on ELISA kit (Bethyl Laboratories) to detect the level of the NP-specific IgM and IgG1 in immunised mice. Anti-dsDNA IgG levels of unimmunised mice and chimeric mice were quantified by ELISA as previously described. 25

BM chimeras
BM cells from WT or Ccr2-KO (CD45.2) mice and WT (CD45.1) mice were mixed at a 50:50 ratio. Recipient WT mice (CD45.1) were irradiated with 7 Gy X-ray and injected with 5 × 10 6 mixed cells through caudal vein under aseptic condition. One week before the radiation and 2 weeks after the injection, mice were given water with antibiotics (gentamicin and erythromycin). To verify the B-cell-specific effect of CCR2 deficiency, BM cells from WT or Ccr2-KO mice and μMT mice were mixed at a 20:80 ratio, and then injected into irradiated recipient WT mice. FCM, immunohistochemistry and immunofluorescence were performed 8 weeks later.

Statistical analysis
The normality of all the data was examined and twotailed unpaired Student's t-test was used (Prism 8.0.1, GraphPad Software). Image J software was used for grey quantification of the immunoblotting stripes to compare protein expression levels. In FCM, the statistical analysis of BM subsets or peripheral B-cell subsets was normalised according to the total number of cells in each corresponding tissue. Colocalisation was displayed using the Pearson's correlation coefficient. Data were extracted from at least three individual experiments, the figures shown are representative figures. Error bars were shown as mean (± SD). p < .05 represented significantly difference.

CCR2 deficiency disturbs the peripheral differentiation of B cells
The expression level of CCR2 on BM subsets was as follows: , and the expression level of CCR2 on splenic B subsets was as follows: MZ > transitional type-1 (T1) ≈ transitional type-2 (T2) > follicular (FO) B cell ( Figure S1A and B). CCR2 expression on B1 cells was significantly higher than that on peritoneal cavity B2 cells, no difference in CCR2 expression between B1a and B1b cells was found ( Figure S1C). In BM, CCR2 deficiency had little impact on B-cell development in Ccr2-KO mice (Figure S1D-F). However, by detecting splenic B-cell subsets ( Figure 1A-C), we found that Ccr2-KO mice had lower proportion and number of FO B cells than WT mice ( Figure 1D). For T1, T2, MZ and germinal centre (GC) B cells, no significant difference between WT and Ccr2-KO mice was found ( Figure 1E-H). Besides, there was no difference in peritoneal cavity B2, B1a and B1b cells (Figure S1G-L). To eliminate the potential effect of other immune cells, especially T cells, on the abnormal differentiation in Ccr2-KO mice, irradiated CD45.1 recipient mice were injected with mixed BM cells from WT or KO mice and CD45.1 mice, a decrease of FO B cells in the CD45.2 population verified the inherent decrease of FO B cells in CCR2-deficient mice ( Figure  S2A-E). However, we also found a decrease of FO B cells in CD45.1 population ( Figure S2F and G). The significant IgG deposition in the glomeruli of Ccr2-KO mice accompanied by increased level of anti-dsDNA IgG ( Figure 1I and J) was observed, and there was no difference between male and female mice ( Figure S3A). Besides, Ccr2-KO mice showed more severe lymphocyte infiltration in the lungs ( Figure 1K), but not in liver, kidney and colon ( Figure S3B). Further, irradiated WT mice were injected with mixed BM cells from WT or Ccr2-KO mice and μMT mice. The decreased proportion and cell number of FO B cells and the decreased number of other peripheral B-cell subsets confirmed the B-cell-specific effect of CCR2 on mice ( Figure  S4A-N). Similarly, glomerular IgG deposition and lung , the red arrows indicated lymphocytic infiltration around blood vessels. Error bars were shown as mean (± SD). Each symbol represents a mouse. *p < .05, **p < .01, ns: no statistical significance lymphocytic infiltration were more severe in Ccr2-KO chimeras than WT chimeras ( Figure S4O-Q). Collectively, CCR2 plays a crucial part in regulating B-cell peripheral differentiation and maintaining the integrity of peripheral autoimmunity.

BCR proximal signalling is upregulated in Ccr2-KO mice
Upon stimulation with sAg, the colocalisation between BCR and pCD19 in Ccr2-KO B cells was significantly increased at 5 min and gradually decreased to the 30 min time-point when BCR is internalised (Figures 2A and B  and S5A). Notably, although the colocalisation between pCD19 and BCR after 10 min of stimulation decreased in Ccr2-KO B cells, the aggregation of pCD19 remained at higher levels than that in WT B cells. This indicates that CCR2 deficiency may affect not only the temporal but also the spatial organisation of CD19. The levels of pCD19, pCD79a and pSyk in B cells were increased under CCR2 deficiency ( Figures 2C and S5B-D). Next, we evaluated the total BCR signaling level-pY, the level of phosphorylated BTK and BCR signaling negative regulator SHIP-1. Compared to WT B cells, the colocalisation between pY and BCR in CCR2-deficient B cells was increased from 5 to 10 min after stimulation ( Figure 2D and E), and that between pBTK and BCR was increased at 5 min after stimulation ( Figures 2F and S5E). The expression of pY (Figures 2G and S5F and G) and pBTK (Figures 2H and S5H and I) in Ccr2-KO B cells was increased. Similarly, the level of pSHIP-1 in Ccr2-KO B cells also increased after 5 and 10 min of activation ( Figure S5J and K), and its colocalisation with BCR peaked at 10 min ( Figure 2I). A higher expression of pSHIP-1 in Ccr2-KO B cells was also verified by immunoblotting (Figures 2J and S5L). Syk inhibitor R406 was used to block BCR signal, and the elevated pBTK and pSHIP-1 caused by CCR2 deficiency were rescued ( Figure 2K).
To sum up, BCR proximal signalling molecules were upregulated in the absence of CCR2, accompanied by enhanced intracellular Ca 2+ activity after BCR activation.

Energy metabolism mediated by PI3K/AKT/mTORC1 is enhanced in CCR2-deficient B cells
One of the subsequent effects of BCR activation is metabolic activity. Cell differentiation-associated molecule mTOR complex 1 (mTORC1) can mediate HIF-1 expression to consequently influence the cell energy metabolism process via the PI3K/AKT pathway. 27 Following sAg stimulation and specific Abs incubation, the expression of phosphorylated PI3K (pPI3K), pAKT, pS6, pmTOR and AKT distal glucose metabolism-related transcriptional factor (pFOXO1) was increased in CCR2-deficient B cells ( Figures 3A and S6A-E). Second, similar to the level of BCR signal blocked by Syk inhibitor R406 (Figure 3B), the expression of pPI3K, pAKT, pFOXO1, pS6 and the proximal signalling molecules pSHIP-1, pBTK and pCD19 in CCR2-deficient B cells incubated with rapamycin, an mTORC1 inhibitor, could be rescued to the level of WT mice ( Figure 3C). This indicates that mTORC1 is necessary for CCR2-regulated B-cell metabolism. In addition, following stimulation with F(ab') 2 -Ig (M + G), seahorse assay showed that Ccr2-KO B cells had higher ATP production levels and maximal respiratory capacity than WT B cells ( Figure 3D), however, the glycolysis ability of Ccr2-KO B cells was decreased ( Figure 3E). Besides, upon in vitro LPS, C P G or F(ab') 2 -Ig (M + G) stimulation, Ccr2-KO B cells showed a faster proliferation ( Figure 3F), although no significant change in the apoptosis was found ( Figure 3G and H).
BCR induces heightened aerobic glycolysis by promoting glucose and oxygen utilisation, and B-cell activation drives Myc-dependent upregulation of glucose transporter 1 and HIF-1α-mediated upregulation of oxygen transport. 28 In immunoblotting analysis, Ccr2-KO B cells exhibited increased levels of HIF-1α and c-MYC ( Figure 3I).
Taken together, in the metabolic activity after BCR activation, CCR2 mainly interacts with PI3K/AKT/mTORC1 pathway to regulate the B cells metabolic signal.

CCR2 deficiency promotes MST1-regulated F-actin accumulation and BCR internalisation
During sAg-induced BCR clustering, cortex actin is detached from the membrane, and F-actin accumulates near BCR microclusters in a polarised manner. 5,6 The Factin levels observed in activated CCR2-deficient B cells were consistently higher than that in WT B cells ( Figure 4A and S7A), as well as the colocalisation between F-actin and BCR after 5 min of stimulation ( Figure 4B). Similarly, the level of pWASP, the actin nucleation-promoting factor and the colocalisation between pWASP and BCR in sAg stimulated Ccr2-KO B cells were also increased ( Figure 4C and S7B). Phosphoflow cytometry demonstrated the expression of F-actin and pWASP increased at 10 and 5 min under sAg stimulation, respectively ( Figure S7C and D). Additionally, more detailed morphologic changes of B cells were obtained by SEM ( Figure 4D). The number and length of filopodia were remarkably elevated in Ccr2-KO B cells upon sAg stimulation ( Figure 4E).
Compared with CFm, TIRFm has a higher resolution when taking spatial pictures of B cells at the moment of contact with mAg. The level of F-actin in the contact zone of KO B cells increased compared with WT B cells after 3-5 min of mAg stimulation ( Figure 4F Figure 4H). Moreover, these changes were accompanied by the increased accumulation of pCD19 in CCR2-deficient B cells at 3 and 5 min of stimulation ( Figure 4I-K), and pY, pBTK and pSHIP-1 were highly recruited in CCR2-deficient B cells ( Figure S7E-I). MST1 regulates cytoskeletal microtubule dynamics and promotes F-actin polymerisation 29 and also modulates BCR to induce actin remodelling by attaching WASP. 30 In the absence of CCR2, the levels of pMST1, pWASP and DOCK8 were increased after sAg stimulation ( Figures 4L and S7J-M). After pre-treatment with MST1 inhibitor, XMU-MP-1, the increased expression of pmTOR, pAKT, pS6 and DOCK8 in Ccr2-KO B cells were rescued ( Figure 4M). And also, pre-treatment with rapamycin can rescue the elevation of DOCK8 and pWASP levels in CCR2deficient B cells ( Figure 4N), suggesting the suppression of mTORC1 in CCR2-deficient B cells can influence the DOCK8/WASP axis. Taken together, CCR2 deletion promoted actin accumulation and BCR internalisation through the activity of MST1/mTORC1/DOCK8/WASP axis.

CCR2 depletion triggers the activation of STAT1 to enhance BCR signalling
During BCR signal transduction, intracellular signal transduction can be connected with intranuclear signal regulation through activation of transcription factors. 31 CCR2 is associated with the activity of the JAK/STAT pathway and its downstream transcriptional factors, 32 and the inhibition on the CCL2/CCR2 axis can affect the activity of NF-κB. 33 Upon sAg stimulation, the interaction between pSTAT1 and BCR in CCR2-deficient B cells significantly increased within 10 min after stimulation ( Figures 5A  and B and S8A). And the interaction between pNF-κB or pSTAT5 and BCR was also higher in CCR2-deficient B cells at 5 min ( Figure S8B-G). Increased expression of pSTAT1, pSTAT5 and pNF-κB in CCR2-deficient B cells was also found at the protein level ( Figures 5C  and S8H-K).
Previous study showed that MST1 regulates the AKT/mTOR pathway 34 and mediates the activation of JAK/STAT downstream transcription factors. 35 After pre-treated with STAT1 inhibitor, fludarabine, the levels of pAKT, pS6, DOCK8, pNF-κB, and pIKKB in Ccr2-KO B cells were rescued ( Figure 5D). Further, following pretreatment with XMU-MP-1 or rapamycin, the upregulated expression of pSTAT1 and pSTAT5 in CCR2-deficient B cells was rescued to the similar level in WT B cells ( Figure 5E and F). Similarly, elevated levels of pNF-κB and pSTAT5 in Ccr2-KO B cells can be rescued by R406 ( Figure 5G). Mechanistically, according to the variation trend of molecule signal after the inhibition of MST1, mTORC1 and STAT1 pathways, we suggested that in CCR2-deficient mice, a sequential activity of MST1/mTORC1/STAT1 is involved in the regulation on BCR signalling.

CCR2 deficiency attenuates the response of mice to T-cell-dependent antigens
Based on the upregulation of BCR-related signalling molecules by CCR2 deletion, the impact of CCR2 on B-cell function were explored by immunising WT and Ccr2-KO mice with T-cell-dependent antigen. After immunisation with 40 μg NP-KLH, spleens of mice were harvested to determine the peripheral B-cell subsets and antibodysecreting cells. In contrast to unimmunised condition, immunised Ccr2-KO mice exhibited increased number of FO B, MZ B, T1 and T2 cells than that in WT mice ( Figure 6A-D). There was no alteration in GC B cells before and after immunisation ( Figure 6E). Moreover, the proportion and/or number of plasmablasts (PBC) and plasma cells (PC) were reduced in Ccr2-KO mice ( Figure 6F-I).
We also noticed a downward trend in the proportion and number of memory B cells (MBC) ( Figure 6J).
Besides, enlarged lymphatic follicles structures in the spleens of immunised Ccr2-KO mice were observed ( Figure 6K), and NP-specific IgM level in CCR2-deficient mice was significantly reduced ( Figure 6L). In summary, the absence of CCR2 was sufficient to induce immune dysfunction in mice.

CCR2 expression is decreased in PBMC of SLE patients
Lastly, we investigated the level of CCR2 in the peripheral blood of SLE patients. First, by using GEO database platform of NCBI, we analysed the level of CCR2 in the peripheral blood of SLE patients and HCs in currently available microarray data sets of relatively large sample size. For the whole peripheral blood, data from GSE61635 set (SLE n = 99, HC n = 30) 36 showed that the level of CCR2 in the peripheral blood of SLE patients was lower than that of HCs (p = .0029) ( Figure 7A). For PBMC, Error bars were shown as mean (± SD). Each symbol represents one patient. *p < .05, **p < .01, ****p < .0001 data from GSE121239 set (SLE n = 292, HC n = 20) 37,38 also showed a decreased level of CCR2 in PBMC of SLE patients, compare to HCs (p = .0323) ( Figure 7B). Then, we performed bioinformatics analysis on the expression of CCR2 in the collected outpatient samples. RNA sequencing showed that there was a downward trend in terms of the expression of CCR2 in PBMC, PW and PG of SLE patients ( Figure 7C), compared to HCs, although not statistically significant. In addition, CCR2 expression also varied among the three types of cells, with the highest expression of CCR2 in PBMC ( Figure 7D). Meanwhile, we detected the level of ccr2 by RT-PCR, results verified a decreased expression of ccr2 in PBMC of SLE patients ( Figure 7E). Also, in PBMC or peripheral blood B cells of SLE patients, western blotting and FCM showed a decrease of CCR2 expression ( Figure 7F and G). Thus, CCR2 expression was reduced at multiple levels in SLE patients.

DISCUSSION
So far, the interaction between CCR2 and BCR signalling has been poorly studied. In this study, we innovatively illustrated the phenotype and biological changes of B cells in CCR2-deficient mice. CCR2 deficiency upregulates key BCR proximal signalling, increases MST1 level acting on mTORC1 and STAT1, thereby promoting actin remodelling, B-cell metabolic proliferation and B cells downstream transcriptional signalling. B-cell immune response in CCR2-deficient mice appeared to be weakened and IgM levels decreased. In addition, the expression of CCR2 on the peripheral blood B cells of SLE patients was lower than that of healthy patients. CCR2 is involved in both immune homeostasis and immune dysregulation. The entry of monocytes from BM into the peripheral circulation requires the chemotactic function of CCR2. 39 SLE susceptibility gene IRF5 can regulate the expression of CCR2 in Ly6C monocytes, thus affecting the migration of monocytes to the peritoneal cavity. 40 In addition, CCR2 deficiency affects not only monocytes or macrophages and T-cell infiltration in lupus kidney but also systemic T-cell response in lupus mice. 41 CCR2 expression is significantly elevated in plasma cells of bronchoalveolar lavage fluid of patients infected with SARS-CoV-2. 42 MHC-II expression on CCR2 negative myocardial macrophages can be regulated by B cells, 43 and inhibition of B cells in CCR2-deficient arthritic mice alleviates arthritic symptoms. 44 B-cell proliferation plays an important role in the pathogenesis of SLE, B-cell components are severely disrupted in lupus patients. 45 Accelerated apoptosis and dysfunction of atypical memory B cells have been reported in SLE patients. 46 Cyclophosphamide can treat SLE by targeting activated B cells that are undergoing proliferation, 47 monoclonal antibodies to CD20 and CD22 have been used to deplete B cells in SLE patients and lupus mice. 48 In this study, the reduced expression of CCR2 in peripheral B cells of SLE patients was noticed, and the metabolic activity of B cells in CCR2-deficient mice was significantly increased, thus promoting the development of autoimmunity. This was also reflected by the significant deposition of IgG immune complex in the glomeruli and increased anti-dsDNA IgG level in CCR2-deficient mice. MTORC1 activation has become an important pathologic pathway in SLE and other autoimmune diseases. Blocking mTORC1 pathway can inhibit T-bet expressing B-cell generation, 46 lymphocyte activation and progression of lupus nephritis of SLE prone mice. 49 Inhibition of the mTOR pathway also reduces the production of anti-dsDNA antibodies produced by B-cell immune disorders. 50 In addition, inhibition of the JAK2/STAT1 pathway in lupus nephritis mice can alleviate renal function damage and immune complex deposition and reduce the level of proteinuria and anti-dsDNA IgG. 51 In this study, both mTORC1 and STAT1 signalling were enhanced in CCR2-deficient mice, promoting B-cell metabolism and transcriptional signalling, thus suggesting the role of this mechanism in autoimmunity.
CCL2 has been confirmed as a negative regulator of BCR signal. 24 In this study, we complemented the role of CCR2, thus linking CCL2-CCR2 and clarifying the role of this axis in BCR signalling. Defects in the CCL2-CCR2 signalling axis upregulate B-cell signalling, actin remodelling and B-cell metabolic activity in mice. BCR signal strength can affect the tendency of B cells to differentiate into FO or MZ groups. 52 CCL2 deficiency leads to decreased MZ B cells and increased spontaneous germinal centres, while CCR2 deficiency leads to fewer FO B cells. In adaptive immune response, FO B cells interact with helper T cells to form GCs, produce high-affinity antibodies to against foreign antigens, or differentiate into memory B cells to prevent the reinfection. 3,53 In the absence of CCR2, the reduction of FO B cells leads to a reduction in the proportion or number of plasma cells and plasmablasts under T-cell-dependent antigen stimulation. Biological signals tend to remain within a range to maintain immune homeostasis, abnormalities in either receptors or ligands can trigger certain thresholds leading to immune disorders. Overenhanced B-cell signalling in CCR2deficient mice reduced antibody secretion during immune response.
In the process of B-cell development and immune response, BCR signals activate numerous upstream and downstream signalling pathways in response to chemokines and receptors, thus achieving accurate and effective regulation. The regulation of CCR2 on B cells in the pathogenesis of SLE needs further study, finding targeted drugs from corresponding signalling pathways deserves to take a place in the treatment of autoimmune diseases.

CONCLUSIONS
CCR2 regulates BCR signalling to affect the biological activity of B cells, involving multiple signalling pathways dominated by MST1, mTORC1 and STAT1. It is worth investigating the effect of CCR2 in autoimmune diseases, so as to identify potential therapeutic targets.

A C K N O W L E D G E M E N T S
We would like to thank Professor Michael R. Gold from Department of Microbiology and Immunology, University of British Columbia for the helpful advice on our study.

C O N F L I C T O F I N T E R E S T
The authors declare no competing financial interests.